Mirna fingerprint in the diagnosis of prostate cancer

ABSTRACT

MicroRNAs (miRNA) are a recently discovered class of small non-coding RNAs (17-14 nucleotides). Due to their function as regulators of gene expression they play a critical role both in physiological and in pathological processes, such as cancer. The present invention provides novel methods for diagnosing a state of health based on the determination of specific miRNAs that have altered expression levels in different conditions, e.g. disease states compared to healthy controls.

BACKGROUND OF THE INVENTION

MicroRNAs (miRNA) are a recently discovered class of small non-codingRNAs (17-14 nucleotides). Due to their function as regulators of geneexpression they play a critical role both in physiological and inpathological processes, such as cancer (Calin and Croce 2006;Esquela-Kerscher and Slack 2006; Zhang, Pan et al. 2007; Sassen, Miskaet al. 2008).

There is increasing evidence that miRNAs are not only found in tissuesbut also in human blood both as free circulating nucleic acids and inmononuclear cells. A recent proof-of-principle study demonstrated miRNAexpression pattern in pooled blood sera and pooled blood cells, both inhealthy individuals and in cancer patients including patients with lungcancer (Chen, Ba et al. 2008). In addition, a remarkable stability ofmiRNAs in human sera was recently demonstrated (Chen, Ba et al. 2008;Gilad, Meiri et al. 2008). These findings make miRNA a potential toolfor diagnostics for various types of diseases based on blood analysis.

Thus, although various markers have been proposed to indicate specifictypes of disorders such as prostate cancer, Wilms' tumour or COPD(Chronic obstructive pulmonary disease) there is still a need for moreefficient and effective methods and compositions for the diagnosis ofdiseases.

SUMMARY OF THE INVENTION

The present invention provides novel methods for diagnosing diseasesbased on the determination of specific miRNAs that have alteredexpression levels in disease states compared to healthy controls oraltered expression levels in a condition 1 (biological state or healthstate 1) compared to a condition 2 (biological state or health state 2).The disease is particularly selected from prostate cancer.

Definitions

miRNA

microRNAs (miRNA or μRNA) are single-stranded RNA molecules of ˜21-23nucleotides in length, which regulate gene expression. miRNAs areencoded by genes from whose DNA they are transcribed but miRNAs are nottranslated into protein (i.e. they are non-coding RNAs). The genesencoding miRNAs are much longer than the processed mature miRNAmolecule; miRNAs are first transcribed as primary transcripts orpri-miRNA with a cap and poly-A tail and processed to short,70-nucleotide stem-loop structures known as pre-miRNA in the cellnucleus. This processing is performed in animals by a protein complexknown as the Microprocessor complex, consisting of the nuclease Droshaand the double-stranded RNA binding protein Pasha. These pre-miRNAs arethen processed to mature miRNAs in the cytoplasm by interaction with theendonuclease Dicer, which also initiates the formation of theRNA-induced silencing complex (RISC). When Dicer cleaves the pre-miRNAstem-loop, two complementary short RNA molecules are formed, but onlyone is integrated into the RISC. This strand is known as the guidestrand and is selected by the argonaute protein, the catalyticallyactive RNase in the RISC, on the basis of the stability of the 5′ end.The remaining strand, known as the miRNA*, anti-guide or passengerstrand, is degraded as a RISC substrate. Therefore the miRNA*s arederived from the same hairpin structure like the “normal” miRNAs. So ifthe “normal” miRNA is then later called the “mature miRNA” or “guidedstrand”, the miRNA* is the passenger strand.

miRNA* (See Also Above “miRNA”)

The miRNA*s, also known as the anti-guide or passenger strand, aremostly complementary to the guide strand, but there are usuallysingle-stranded overhangs on each end, there is usually one or a fewmispairs and there are sometimes extra or missing bases causingsingle-stranded “bubbles. The miRNA*s are likely to act in a regulatoryfashion as the miRNAs. It is understood that according to the presentinvention the term “miRNA” also includes the term “miRNA*”.

miRBase

A well established repository of validated miRNAs is the miRBase. ThemiRBase (www.mirbase.org) is a searchable database of published miRNAsequences and annotation. Each entry in the miRBase Sequence databaserepresents a predicted hairpin portion of a miRNA transcript (termed mirin the database), with information on the location and sequence of themature miRNA sequence (termed miR). Both hairpin and mature sequencesare available for searching and browsing, and entries can also beretrieved by name, keyword, references and annotation. All sequence andannotation data are also available for download.

miRNA-(expression) Profile or miRNA Fingerprint

A miRNA-Profile represents the collection of expression levels of aplurality of miRNAs, therefore it is a quantitative measure ofindividual miRNA expression levels. Hereby, each miRNA is represented bya numerical value. The higher the value of an individual miRNA thehigher is the expression level of this miRNA. A miRNA-profile isobtained from the RNA of a biological sample. The are varioustechnologies to determine a miRNA-Profile, e.g. microarrays, RT-PCR,Next Generation Sequencing. As a starting material for analysis, RNA ortotal-RNA or any fraction thereof can be used. The plurality of miRNAsthat are determined by a miRNA-profile can range from a selection of oneup to all known miRNAs.

Pre-determined Set of miRNAs or miRNA Signature

The pre-determined set of miRNAs or miRNA signature is understood in thepresent invention as a fixed defined set of miRNAs which is able todifferentiate between a condition 1 and another condition 2. e.g. whencondition 1 is lung cancer and condition 2 is normal control, thecorresponding pre-determined set of miRNAs is able to differentiatebetween a samples derived from a lung cancer patient or a normal controlpatient. Alternatively, condition 1 is lung cancer and condition 2 ismultiple sclerosis, the corresponding pre-determined set of miRNAs isable to differentiate between a lung cancer patient and a multiplesclerosis patient. In order to be able to perform the sample analysis itis required that, e.g. on the matrix that will be used to determine amiRNA profile, these fixed defined set of miRNAs have to be representedby capture probes that are defined by the predetermined set of miRNAs.For example, when the predetermined set of miRNAs for diagnosing lungcancer from healthy controls consists of 25 miRNAs, probes capable fordetecting these 25 miRNAs have to be implemented for performing thediagnostic analysis.

Common miRNA Signature Profile

A common miRNA signature profile is understood in the present inventionas a non-fixed defined set of miRNAs or non-coding RNAs which is able todifferentiate between a condition 1 and another condition 2. The commonmiRNA or non-coding RNA signature profile is calculated “on-the-fly”from a plurality of miRNA-profiles that are stored, e.g. in database.The common miRNA signature profile which is able to differentiatebetween a condition 1 and another condition 2 is changing as soon as annew profile is added to the database which is relevant to either tostate of health 1 or another condition 2. In this respect it isdifferent from a predetermined set of miRNAs (see above). Furthermore,the basis for generating the common miRNA signature profile—hence themiRNA profiles stored in the database—is generated from capture probes,e.g. on a matrix that is representing as much as possible differentcapture probes for detecting as much as possible, ideally all known,miRNAs.

Non-coding RNA

A non-coding RNA (ncRNA) is a functional RNA molecule that is nottranslated into a protein. Less-frequently used synonyms arenon-protein-coding RNA (npcRNA), non-messenger RNA (nmRNA), smallnon-messenger RNA (snmRNA), functional RNA (fRNA). The term small RNA(sRNA) is often used for bacterial ncRNAs. The DNA sequence from which anon-coding RNA is transcribed as the end product is often called an RNAgene or non-coding RNA gene.

Non-coding RNA genes include highly abundant and functionally importantRNAs such as transfer RNA (tRNA) and ribosomal RNA (rRNA), as well asRNAs such as snoRNAs, microRNAs, siRNAs and piRNAs and the long ncRNAsthat include examples such as Xist and HOTAIR (see here for a morecomplete list of ncRNAs). The number of ncRNAs encoded within the humangenome is unknown, however recent transcriptomic and bioinformaticstudies suggest the existence of thousands of ncRNAs. Since most of thenewly identified ncRNAs have not been validated for their function, itis possible that many are non-functional.

Condition

A condition (biological state or health state or state of health) isunderstood in the present invention as status of a subject that can bedescribed by physical, mental or social criteria. It includes as wellso-called “healthy” and “diseased” conditions, therefore it is notlimited to the WHO definition of health as “a state of completephysical, mental, and social well-being and not merely the absence ofdisease or infirmity.” but includes disease and infirmity. For thedefinition of diseases comprised, e.g. by the conditions of the presentinvention, it is referred to the international classification ofdiseases (ICD) of the WHO (http://www.who.int/classifications/icd/en/index.html). When 2 or more conditions are compared according to thepresent invention, it is understood that this is possible for allconditions that can be defined and is not limited to a comparison of adisease versus healthy and extends to multi-way comparisons. Examplesfor comparison are, but not limited to:

Pairwise Comparisons:

-   -   lung cancer vs. healthy control, pancreatic cancer vs. healthy        control    -   lung cancer vs. pancreatic cancer, lung cancer vs. multiple        sclerosis    -   lung cancer WHO grade 1 vs. lung cancer WHO grade 2    -   lung cancer WHO grade 1 metastasing vs. lung cancer WHO grade 1        non-metastasing    -   Morbus Crohn vs. collitis    -   Pancreatic cancer vs. pancreatitis

Multi-Way Comparisons:

-   -   Lung cancer vs. pancreatic cancer vs. multiple sclerosis    -   Pancreas cancer vs. pancreatitis vs. lung cancer WHO grade 1        non-metastasing

Prostate Cancer

Prostate cancer is a form of cancer that develops in the prostate, agland in the male reproductive system. The cancer cells may metastasize(spread) from the prostate to other parts of the body, particularly thebones and lymph nodes. Prostate cancer may cause pain, difficulty inurinating, problems during sexual intercourse, or erectile dysfunction.Other symptoms can potentially develop during later stages of thedisease.

Rates of detection of prostate cancers vary widely across the world,with South and East Asia detecting less frequently than in Europe, andespecially the United States. Prostate cancer tends to develop in menover the age of fifty and although it is one of the most prevalent typesof cancer in men, many never have symptoms, undergo no therapy, andeventually die of other causes. This is because cancer of the prostateis, in most cases, slow-growing, symptom free and men with the conditionoften die of causes unrelated to the prostate cancer, such asheart/circulatory disease, pneumonia, other unconnected cancers, or oldage. Many factors, including genetics and diet, have been implicated inthe development of prostate cancer. The presence of prostate cancer maybe indicated by symptoms, physical examination, prostate specificantigen (PSA), or biopsy. There is controversy about the accuracy of thePSA test and the value of screening. Suspected prostate cancer istypically confirmed by taking a biopsy of the prostate and examining itunder a microscope. Further tests, such as CT scans and bone scans, maybe performed to determine whether prostate cancer has spread.

Treatment options for prostate cancer with intent to cure are primarilysurgery, radiation therapy, and proton therapy. Other treatments, suchas hormonal therapy, chemotherapy, cryosurgery, and high intensityfocused ultrasound (HIFU) also exist, depending on the clinical scenarioand desired outcome.

The age and underlying health of the man, the extent of metastasis,appearance under the microscope, and response of the cancer to initialtreatment are important in determining the outcome of the disease. Thedecision whether or not to treat localized prostate cancer (a tumourthat is contained within the prostate) with curative intent is a patienttrade-off between the expected beneficial and harmful effects in termsof patient survival and quality of life.

A “biological sample” in terms of the invention means a sample ofbiological tissue or fluid. Examples of biological samples are sectionsof tissues, blood, blood fractions, plasma, serum, urine or samples fromother peripheral sources. or cell cultures, cell colonies of even singlecells, or a collection of single cells. Furthermore, also pools ormixture of the above mentioned samples may be employed. A biologicalsample may be provided by removing a sample of cells from a subject, butcan also be provided by using a previously isolated sample. For example,a tissue sample can be removed from a subject suspected of having adisease by conventional biopsy techniques. In a preferred embodiment, ablood sample is taken from the subject. In one embodiment, the blood ortissue sample is obtained from the subject prior to initiation ofradiotherapy, chemotherapy or other therapeutic treatment. According tothe invention, the biological sample preferably is a blood or a serumsample. Further, it is also preferred to use blood cells, e.g.erythrocytes, leukocytes or thrombocytes.

A biological sample from a patient means a sample from a subjectsuspected to be affected by a disease. As used herein, the term“subject” refers to any mammal, including both human and other mammals.Preferably, the methods of the present invention are applied to humansubjects.

Subject-matter of the invention is a method for diagnosing a disease,comprising the steps

-   -   (a) determining an expression profile of a predetermined set of        miRNAs in a biological sample from a patient (or subject); and    -   (b) comparing said expression profile to a reference expression        profile,        wherein the comparison of said determined expression profile to        said reference expression profile allows for the diagnosis of        the disease.

In step (a) of the above method of the invention, an expression profileof a predetermined set of miRNAs is determined. The determination may becarried out by any convenient means for determining nucleic acids. Forexpression profiling, qualitative, semi-quantitative and preferablyquantitative detection methods can be used. A variety of techniques arewell known to those of skill in the art. In particular, thedetermination may comprise nucleic acid hybridization and/or nucleicacid amplification steps.

Nucleic acid hybridization may for example be performed using a solidphase nucleic acid biochip array, in particular a microarray, or in situhybridization. The miRNA microarray technology affords the analysis of acomplex biological sample for all expressed miRNAs. Nucleotides withcomplementarity to the corresponding miRNAs are spotted on coatedcarriers or are fabricated by in-situ synthesis methods on a carrier.Preferably, miRNAs isolated from the sample of interest are notlabelled, e.g. before hybridization of the miRNAs to the complementarysequences on the carrier and the resulting signal indicating theoccurrence of a distinct miRNA is generated by incorporation of adetectable label (e.g. biotin, fluorescent dye) by means of an enzymereaction.

According to another embodiment of the invention, miRNAs isolated fromthe sample of interest are labelled, e.g. fluorescently labelled, sothat upon hybridization of the miRNAs to the complementary sequences onthe carrier the resulting signal indicates the occurrence of a distinctmiRNA. On one miRNA microarray, preferably at least the wholepredetermined set of miRNAs can be analyzed.

Further, quantitative real-time polymerase chain reaction (RT-PCR) canbe used to detect miRNAs even at very low abundance.

Alternative methods for obtaining expression profiles may also containsequencing, next generation sequencing or mass spectroscopy.

The predetermined set of miRNAs in step (a) of the above method of theinvention depends on the disease to be diagnosed. The inventors foundout that single miRNA biomarkers lack sufficient accuracy, specificityand sensitivity, and therefore it is preferred to analyze more complexmiRNA expression patterns, so-called miRNA signatures. The predeterminedset of miRNAs comprises one or more, preferably a larger number ofmiRNAs (miRNA signatures) that are differentially regulated in samplesof a patient affected by a particular disease compared to healthycontrols. Alternatively, the disease can also be compared to any otherdefined condition (e.g. another disease).

The expression profile determined in the above step (a) is subsequentlycompared to a reference expression profile or to a plurality ofreference profiles in the above step (b). The reference expressionprofile is the expression profile of the same set of miRNAs in abiological sample originating from the same source as the biologicalsample from a patient but obtained from a healthy subject. Preferably,both the reference expression profile and the expression profile of theabove step (a) are determined in a blood or serum sample or in a sampleof erythrocytes, leukocytes and/or thrombocytes. It is understood thatthe reference expression profile is not necessarily obtained from asingle healthy subject but may be an average expression profile of aplurality of healthy subjects. It is preferred to use a referenceexpression profile obtained from a person of the same gender, and asimilar age as the patient.

The above method of the invention is suitable for diagnosing anydiseases for which a differential expression of miRNAs compared tohealthy controls or other diseases exists. In particular, the method maybe used for diagnosing cancer including bladder cancer, brain cancer,breast cancer, colon cancer, endometrium cancer, gastrointestinalstromal cancer, glioma, head- and neck cancer, kidney cancer, leukemia,liver cancer, lung cancer, lymph node cancer, melanoma, meninges cancer,ovarian cancer, pancreas cancer, prostate cancer, sarcoma, stomachcancer, testicular cancer, thyroid cancer, thymus cancer and Wilms'tumour or COPD. The diagnosis may comprise determining type, rate and/orstage of cancer. The course of the disease and the success of therapysuch as chemotherapy may be monitored. The method of the inventionprovides a prognosis on the survivor rate and enables to determine apatient's response to drugs.

In addition to cancer, also different types of diseases may be diagnosedby means of the above method of the invention, if the disease state iscorrelated with a differential expression of miRNAs compared to ahealthy control. For example the disease may be Alzheimer's disease,multiple sclerosis, melanoma, Morbus Crohn and cardiovascular diseases.The inventors found out that also these diseases are correlated with aspecific expression profile of miRNAs.

The inventors succeeded in developing a generally applicable approach toarrive at miRNA signatures that are correlated with a particulardisease. In more detail, the following steps are accomplished:

1. miRNAs are extracted from a biological sample of a patient,preferably a blood or serum or urine sample or a sample comprisingerythrocytes, leukocytes or thrombocytes, using suitable kits /purification methods. From these samples preferably the RNA-fraction isused for analysis.

2. The respective samples are measured using experimental techniques.These techniques include but are not restricted to:

-   -   Array based approaches    -   Real time quantitative polymerase chain reaction    -   Sequencing    -   Next Generation Sequencing    -   Mass Spectroscopy

3. Mathematical approaches are applied to gather information on thevalue and the redundancy of single biomarkers. These methods include,but are not restricted to:

-   -   basic mathematic approaches (e.g. Fold Quotients, Signal to    -   Noise ratios, Correlation)    -   statistical methods as hypothesis tests (e.g. t-test,        Wilcoxon-Mann-Whitney test), the Area under the Receiver        operator Characteristics Curve    -   Information Theory approaches, (e.g. the Mutual Information,        Cross-entropy)    -   Probability theory (e.g. joint and conditional probabilities)    -   Combinations and modifications of the previously mentioned        examples

4. The information collected in 3) are used to estimate for eachbiomarker the diagnostic content or value. Usually, however, thisdiagnostic value of only one biomarker is too small to get a highlyaccurate diagnosis with accuracy rates, specificities and sensitivitiesbeyond the 90% barrier. Please note that the diagnostic content for ourmiRNAs can be found in the tables in FIGS. 2 and 5. These tablesincludes the miRNAs with the sequences, and the significance value ascomputed by a t-test and further statistical measures.

5. Thus statistical learning/machinelearning/bioinformatics/computational approaches are applied to definesubsets of biomarkers that are tailored for the detection of diseases.These techniques includes but are not restricted to

-   -   Wrapper subset selection techniques (e.g. forward step-wise,        backward step-wise, combinatorial approaches, optimization        approaches)    -   Filter subset selection methods (e.g. the methods mentioned in        3)    -   Principal Component Analysis    -   Combinations and modifications of such methods (e.g. hybrid        approaches)

6. The diagnostic content of each detected set can be estimated bymathematical and/or computational techniques to define the diagnosticinformation content of subsets.

7. The subsets, detected in step 5, which may range from only a smallnumber (at least two) to all measured biomarkers is then used to carryout a diagnosis. To this end, statistical learning/machinelearning/bioinformatics/computational approaches are applied thatinclude but are not restricted to any type of supervised or unsupervisedanalysis:

-   -   Classification techniques (e.g. naïve Bayes, Linear Discriminant        Analysis, Quadratic Discriminant Analysis Neural Nets, Tree        based approaches, Support Vector Machines, Nearest Neighbour        Approaches)    -   Regression techniques (e.g. linear Regression, Multiple        Regression, logistic regression, probit regression, ordinal        logistic regression ordinal Probit-Regression, Poisson        Regression, negative binomial Regression, multinomial logistic        Regression, truncated regression)    -   Clustering techniques (e.g. -means clustering, hierarchical        clustering, PCA)    -   Adaptations, extensions, and combinations of the previously        mentioned approaches

The inventors surprisingly found out that the described approach yieldsin miRNA signatures that provide high diagnostic accuracy, specificityand sensitivity in the determination of diseases.

According to a preferred embodiment of the invention, the disease to bedetermined is prostate cancer.

The inventors succeeded in determining miRNAs that are differentiallyregulated in samples from prostate cancer patients as compared tohealthy controls. A complete overview of all miRNAs that are found to bedifferentially regulated in blood samples of prostate cancer patients isprovided in the tables shown in FIGS. 2 and 5.

In the tables shown in FIGS. 2 and 5, the miRNAs that are found to bedifferentially regulated are sorted in the order of their t-testsignificance. Another method for assessing the significance is tocompute the Mutual information (MI) (Shannon, 1984) which is an adequatemeasure to estimate the overall diagnostic information content of singlebiomarkers (Keller, Ludwig et al., 2006). According to the inventionmutual information is considered as the reduction in uncertainty aboutthe class labels “0” for controls and “1” for tumour samples due to theknowledge of the miRNA expression. The higher the value of the MI of amiRNA, the higher is the diagnostic content of the respective miRNA.

Diagnosis of Prostate Cancer

According to a preferred embodiment of the invention, the disease to bedetermined is prostate cancer. Surprisingly, the inventors found outthat miRNAs are differentially regulated in samples from prostate cancerpatients as compared to healthy controls. A complete overview of allmiRNAs that are found to be differentially regulated in blood samples ofprostate cancer patients is provided in the table shown in FIG. 2 andFIG. 5. In FIG. 2 in total, 241 miRNAs were found to be significantlyderegulated (t-test significance <0.05) in blood cells of prostatecancer patients as compared to the healthy controls.

Preferably, the predetermined set of miRNAs for the diagnosis ofprostate cancer comprises one or more nucleic acids selected from thederegulated miRNAs presented in the tables in FIG. 2 or FIG. 5.

The predetermined set of miRNAs should preferably comprise at least 1,preferably at least 7, 10, 15, 20, 25, 30, 35, 40, 50, 75 or 100 of theindicated nucleic acids. It is particularly preferred to include the100, 75, 50, 40, 35, 30, 25, 20, 15, 10 or at least 7 of the firstmentioned miRNAs according to their order in the tables in FIG. 2 orFIG. 5.

Thus, preferably the predetermined set of miRNAs for the diagnosis ofprostate cancer comprises one or more nucleic acids selected from the241 most deregulated miRNAs.

Preferably, the predetermined set of miRNAs comprises at least 7,preferably at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100 or all ofthe above-indicated nucleic acids.

In a further embodiment the predetermined set of miRNAs for thediagnosis of prostate cancer comprises one or more miRNAs selected fromthe group consisting of hsa-miR-144*, hsa-miR-148a, hsa-miR-519b-5p,hsa-miR-1324, hsa-miR-137, hsa-miR-556-5p, hsa-miR-330-3p.

In a further embodiment the predetermined set of miRNAs for thediagnosis of prostate cancer comprises one or more miRNAs selected fromthe group consisting of hsa-miR-144*, hsa-miR-148a, hsa-miR-519b-5p,hsa-miR-1324, hsa-miR-137, hsa-miR-556-5p, hsa-miR-330-3p,hsa-miR-361-5p, hsa-miR-891b, hsa-miR-767-5p.

In a further embodiment the predetermined set of miRNAs for thediagnosis of prostate cancer comprises one or more miRNAs selected fromthe group consisting of hsa-miR-144*, hsa-miR-148a, hsa-miR-519b-5p,hsa-miR-1324, hsa-miR-137, hsa-miR-556-5p, hsa-miR-330-3p,hsa-miR-361-5p, hsa-miR-891b, hsa-miR-767-5p, hsa-miR-744*,hsa-miR-208b, hsa-miR-548p, hsa-miR-20a*, hsa-miR-195.

In a further embodiment the predetermined set of miRNAs for thediagnosis of prostate cancer comprises one or more miRNAs selected fromthe group consisting of hsa-miR-144*, hsa-miR-148a, hsa-miR-519b-5p,hsa-miR-1324, hsa-miR-137, hsa-miR-556-5p, hsa-miR-330-3p,hsa-miR-361-5p, hsa-miR-891 b, hsa-miR-767-5p, hsa-miR-744*,hsa-miR-208b, hsa-miR-548p, hsa-miR-20a*, hsa-miR-195, hsa-miR-33b,hsa-miR-1283, hsa-miR-519c-5p, hsa-miR-497, hsa-miR-9*.

In a further embodiment the predetermined set of miRNAs for thediagnosis of prostate cancer comprises one or more miRNAs selected fromthe group consisting of hsa-miR-144*, hsa-miR-148a, hsa-miR-519b-5p,hsa-miR-1324, hsa-miR-137, hsa-miR-556-5p, hsa-miR-330-3p,hsa-miR-361-5p, hsa-miR-891 b, hsa-miR-767-5p, hsa-miR-744*,hsa-miR-208b, hsa-miR-548p, hsa-miR-20a*, hsa-miR-195, hsa-miR-33b,hsa-miR-1283, hsa-miR-519c-5p, hsa-miR-497, hsa-miR-9*, hsa-miR-200a,hsa-miR-338-3p, hsa-miR-515-5p, hsa-miR-31*, hsa-miR-551 b*.

In a further embodiment the predetermined set of miRNAs for thediagnosis of prostate cancer comprises one or more miRNAs selected fromthe group consisting of hsa-miR-144*, hsa-miR-148a, hsa-miR-519b-5p,hsa-miR-1324, hsa-miR-137, hsa-miR-556-5p, hsa-miR-330-3p,hsa-miR-361-5p, hsa-miR-891 b, hsa-miR-767-5p, hsa-miR-744*,hsa-miR-208b, hsa-miR-548p, hsa-miR-20a*, hsa-miR-195, hsa-miR-33b,hsa-miR-1283, hsa-miR-519c-5p, hsa-miR-497, hsa-miR-9*, hsa-miR-200a,hsa-miR-338-3p, hsa-miR-515-5p, hsa-miR-31*, hsa-miR-551b*,hsa-miR-518e*, hsa-miR-127-5p, hsa-miR-21*, hsa-miR-216a, hsa-miR-452*.

In a further embodiment the predetermined set of miRNAs for thediagnosis of prostate cancer comprises one or more miRNAs selected fromthe group consisting of hsa-miR-144*, hsa-miR-148a, hsa-miR-519b-5p,hsa-miR-1324, hsa-miR-137, hsa-miR-556-5p, hsa-miR-330-3p,hsa-miR-361-5p, hsa-miR-891 b, hsa-miR-767-5p, hsa-miR-744*,hsa-miR-208b, hsa-miR-548p, hsa-miR-20a*, hsa-miR-195, hsa-miR-33b,hsa-miR-1283, hsa-miR-519c-5p, hsa-miR-497, hsa-miR-9*, hsa-miR-200a,hsa-miR-338-3p, hsa-miR-515-5p, hsa-miR-31*, hsa-miR-551 b*,hsa-miR-518e*, hsa-miR-127-5p, hsa-miR-21*, hsa-miR-216a, hsa-miR-452*,hsa-miR-183*, hsa-miR-500, hsa-miR-1826, hsa-miR-625*, hsa-miR-513b.

In a further embodiment the predetermined set of miRNAs for thediagnosis of prostate cancer comprises one or more miRNAs selected fromthe group consisting of hsa-miR-144*, hsa-miR-148a, hsa-miR-519b-5p,hsa-miR-1324, hsa-miR-137, hsa-miR-556-5p, hsa-miR-330-3p,hsa-miR-361-5p, hsa-miR-891 b, hsa-miR-767-5p, hsa-miR-744*,hsa-miR-208b, hsa-miR-548p, hsa-miR-20a*, hsa-miR-195, hsa-miR-33b,hsa-miR-1283, hsa-miR-519c-5p, hsa-miR-497, hsa-miR-9*, hsa-miR-200a,hsa-miR-338-3p, hsa-miR-515-5p, hsa-miR-31*, hsa-miR-551 b*,hsa-miR-518e*, hsa-miR-127-5p, hsa-miR-21*, hsa-miR-216a, hsa-miR-452*,hsa-miR-183*, hsa-miR-500, hsa-miR-1826, hsa-miR-625*, hsa-miR-513b,hsa-miR-526a, hsa-miR-33a, hsa-miR-1243, hsa-miR-517*, hsa-miR-541.

In a further embodiment the predetermined set of miRNAs for thediagnosis of prostate cancer comprises one or more miRNAs selected fromthe group consisting of hsa-miR-144*, hsa-miR-148a, hsa-miR-519b-5p,hsa-miR-1324, hsa-miR-137, hsa-miR-556-5p, hsa-miR-330-3p,hsa-miR-361-5p, hsa-miR-891b, hsa-miR-767-5p, hsa-miR-744*,hsa-miR-208b, hsa-miR-548p, hsa-miR-20a*, hsa-miR-195, hsa-miR-33b,hsa-miR-1283, hsa-miR-519c-5p, hsa-miR-497, hsa-miR-9*, hsa-miR-200a,hsa-miR-338-3p, hsa-miR-515-5p, hsa-miR-31*, hsa-miR-551 b*,hsa-miR-518e*, hsa-miR-127-5p, hsa-miR-21*, hsa-miR-216a, hsa-miR-452*,hsa-miR-183*, hsa-miR-500, hsa-miR-1826, hsa-miR-625*, hsa-miR-513b,hsa-miR-526a, hsa-miR-33a, hsa-miR-1243, hsa-miR-517*, hsa-miR-541,hsa-miR-217, hsa-miR-621, hsa-miR-518d-5p, hsa-miR-873, hsa-miR-103-as,hsa-miR-450b-5p, hsa-miR-545, hsa-miR-1251, hsa-miR-885-5p, hsa-miR-922.

In a further embodiment the predetermined set of miRNAs for thediagnosis of prostate cancer comprises one or more miRNAs selected fromthe group consisting of hsa-miR-144*, hsa-miR-148a, hsa-miR-519b-5p,hsa-miR-1324, hsa-miR-137, hsa-miR-556-5p, hsa-miR-330-3p,hsa-miR-361-5p, hsa-miR-891 b, hsa-miR-767-5p, hsa-miR-744*,hsa-miR-208b, hsa-miR-548p, hsa-miR-20a*, hsa-miR-195, hsa-miR-33b,hsa-miR-1283, hsa-miR-519c-5p, hsa-miR-497, hsa-miR-9*, hsa-miR-200a,hsa-miR-338-3p, hsa-miR-515-5p, hsa-miR-31*, hsa-miR-551b*,hsa-miR-518e*, hsa-miR-127-5p, hsa-miR-21*, hsa-miR-216a, hsa-miR-452*,hsa-miR-183*, hsa-miR-500, hsa-miR-1826, hsa-miR-625*, hsa-miR-513b,hsa-miR-526a, hsa-miR-33a, hsa-miR-1243, hsa-miR-517*, hsa-miR-541,hsa-miR-217, hsa-miR-621, hsa-miR-518d-5p, hsa-miR-873, hsa-miR-103-as,hsa-miR-450b-5p, hsa-miR-545, hsa-miR-1251, hsa-miR-885-5p, hsa-miR-922,hsa-miR-628-5p, hsa-miR-548f, hsa-miR-802, hsa-miR-25, hsa-miR-423-3p,hsa-miR-522*, hsa-miR-519a*, hsa-miR-455-3p, hsa-miR-1245,hsa-miR-362-5p, hsa-miR-1184, hsa-miR-191, hsa-miR-487a, hsa-miR-216b,hsa-miR-525-5p, hsa-miR-509-3-5p, hsa-miR-27a*, hsa-miR-488*,hsa-miR-1226, hsa-miR-646, hsa-miR-527, hsa-miR-635, hsa-miR-1825,hsa-let-7i*.

Most preferably, the predetermined set of miRNAs comprises those miRNAsthat were most significantly deregulated.

In a further embodiment, the measured miRNA profiles were classifiedusing statistical learning approaches in order to compute accuracy,specificity, and sensitivity for the diagnosis of prostate cancer (seeFIG. 4). The miRNAs that performed best for the diagnosis of prostatecancer according to their accuracy, specificity, and sensitivity are the270 miRNAs shown in Table in

FIG. 2 (entries No. 1-270) leading to an accuracy 82.8%, a specificityof 87.5% and a sensitivity of 71.9%.

The predetermined set of miRNAs for the diagnosis of prostate cancershould preferably comprise at least 7, preferably at least 10, 15, 20,25, 30, 35, 40, 50, 75, 100, preferably all of the known miRNAs,preferably all of the 863 (see FIG. 1, representing the current statusof all known miRNAs in the version 12, 13, and 14 of the miRBaserepository (www.mirbase.org).

Another embodiment of the present invention is a kit for diagnosing adisease, comprising means for determining an expression profile of apredetermined set of miRNAs in a biological sample, in particular in ablood and/or serum sample. Preferably, one or more reference expressionprofiles are also provided which show the expression profile of the sameset of miRNAs in the same type of biological sample, in particular in ablood and/or serum sample, obtained from one or more healthy subjects. Acomparison to said reference expression profile(s) allows for thediagnosis of the disease.

Another preferred embodiment of the present invention is a kit fordiagnosing prostate cancer, comprising means for determining theexpression profile of one or more miRNAs presented in the table in FIG.2, preferably one or more miRNAs selected from the group consisting ofhsa-miR-144*, hsa-miR-148a, hsa-miR-519b-5p, hsa-miR-1324, hsa-miR-137,hsa-miR-556-5p, hsa-miR-330-3p, hsa-miR-361-5p, hsa-miR-891 b,hsa-miR-767-5p, hsa-miR-744*, hsa-miR-208b, hsa-miR-548p, hsa-miR-20a*,hsa-miR-195, hsa-miR-33b, hsa-miR-1283, hsa-miR-519c-5p, hsa-miR-497,hsa-miR-9*, hsa-miR-200a, hsa-miR-338-3p, hsa-miR-515-5p, hsa-miR-31*,hsa-miR-551 b*, hsa-miR-518e*, hsa-miR-127-5p, hsa-miR-21*,hsa-miR-216a, hsa-miR-452*, hsa-miR-183*, hsa-miR-500, hsa-miR-1826,hsa-miR-625*, hsa-miR-513b, hsa-miR-526a, hsa-miR-33a, hsa-miR-1243,hsa-miR-517*, hsa-miR-541, hsa-miR-217, hsa-miR-621, hsa-miR-518d-5p,hsa-miR-873, hsa-miR-103-as, hsa-miR-450b-5p, hsa-miR-545, hsa-miR-1251,hsa-miR-885-5p, hsa-miR-922.

In a preferred embodiment the kit comprises means for determining atleast 7, preferably at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100 orall of the indicated miRNAs. It is particularly preferred to includemeans for determining the 100, 75, 50, 45, 40, 35, 30, 25, 20, 15, 10 orat least 7 first mentioned miRNAs in the order of their diagnosticsignificance as represented by their order in the table in FIG. 2. Thekit for diagnosing prostate cancer is particularly suitable fordiagnosing prostate cancer in a blood and/or serum sample or in a samplecomprising erythrocytes, leukocytes and/or thrombocytes.

The means for determining a predetermined set of miRNAs may for examplecomprise a microarray comprising miRNA-specific oligonucleotide probes.In a preferred embodiment, the microarray comprises miRNA-specificoligonucleotide probes for the detection of miRNAs. Depending on theintended use of the microarray in the diagnosis or prognosis of aparticular disease, probes for detecting different miRNAs may beincluded.

A microarray intended for use in the diagnosis of prostate cancerpreferably comprises miRNA specific oligonucleotide probes for one ormore miRNAs presented in the table in FIG. 2, preferably for one or moremiRNAs selected from the group consisting of hsa-miR-144*, hsa-miR-148a,hsa-miR-519b-5p, hsa-miR-1324, hsa-miR-137, hsa-miR-556-5p,hsa-miR-330-3p, hsa-miR-361-5p, hsa-miR-891b, hsa-miR-767-5p,hsa-miR-744*, hsa-miR-208b, hsa-miR-548p, hsa-miR-20a*, hsa-miR-195,hsa-miR-33b, hsa-miR-1283, hsa-miR-519c-5p, hsa-miR-497, hsa-miR-9*,hsa-miR-200a, hsa-miR-338-3p, hsa-miR-515-5p, hsa-miR-31*, hsa-miR-551b*, hsa-miR-518e*, hsa-miR-127-5p, hsa-miR-21*, hsa-miR-216a,hsa-miR-452*, hsa-miR-183*, hsa-miR-500, hsa-miR-1826, hsa-miR-625*,hsa-miR-513b, hsa-miR-526a, hsa-miR-33a, hsa-miR-1243, hsa-miR-517*,hsa-miR-541, hsa-miR-217, hsa-miR-621, hsa-miR-518d-5p, hsa-miR-873,hsa-miR-103-as, hsa-miR-450b-5p, hsa-miR-545, hsa-miR-1251,hsa-miR-885-5p, hsa-miR-922.

In a preferred embodiment the microarray comprises oligonucleotideprobes for determining at least 7, preferably at least 10, 15, 20, 25,30, 35, 40, 50, 75, 100 or all of the indicated miRNAs. It isparticularly preferred to include oligonucleotide probes for determiningthe most significant miRNAs, which is represented by their order in thetable depicted in FIG. 2.

The microarray can comprise oligonucleotide probes obtained from knownor predicted miRNA sequences. The array may contain differentoligonucleotide probes for each miRNA, for example one containing theactive mature sequence and another being specific for the precursor ofthe miRNA. The array may also contain controls such as one or moresequences differing from the human orthologs by only a few bases, whichcan serve as controls for hybridization stringency conditions. It isalso possible to include viral miRNAs or putative miRNAs as predictedfrom bioinformatic tools. Further, it is possible to include appropriatecontrols for non-specific hybridization on the microarray.

In summary the present invention is composed of the following items:

1. A method of diagnosing a disease, comprising the steps

-   -   (a) determining an expression profile of a predetermined set of        non-coding RNAs, including miRNAs, in a biological sample from a        patient; and    -   (b) comparing said expression profile to a reference expression        profile,    -   wherein the comparison of said determined expression profile to        said reference expression profile allows for the diagnosis of        the disease, wherein the disease is prostate cancer.

2. The method according to any one of item 1, wherein the expressionprofile is determined of non-coding RNAs, including miRNAs selected fromthe group consisting of hsa-miR-99b*, hsa-miR-99b, hsa-miR-99a*,hsa-miR-99a, hsa-miR-98, hsa-miR-96*, hsa-miR-96, hsa-miR-95,hsa-miR-944, hsa-miR-943, hsa-miR-942, hsa-miR-941, hsa-miR-940,hsa-miR-939, hsa-miR-938, hsa-miR-937, hsa-miR-936, hsa-miR-935,hsa-miR-934, hsa-miR-933, hsa-miR-93*, hsa-miR-93, hsa-miR-92b*,hsa-miR-92b, hsa-miR-92a-2*, hsa-miR-92a-1*, hsa-miR-92a, hsa-miR-924,hsa-miR-922, hsa-miR-921, hsa-miR-920, hsa-miR-9*, hsa-miR-9,hsa-miR-892b, hsa-miR-892a, hsa-miR-891 b, hsa-miR-891a, hsa-miR-890,hsa-miR-889, hsa-miR-888*, hsa-miR-888, hsa-miR-887, hsa-miR-886-5p,hsa-miR-886-3p, hsa-miR-885-5p, hsa-miR-885-3p, hsa-miR-877*,hsa-miR-877, hsa-miR-876-5p, hsa-miR-876-3p, hsa-miR-875-5p,hsa-miR-875-3p, hsa-miR-874, hsa-miR-873, hsa-miR-802, hsa-miR-770-5p,hsa-miR-769-5p, hsa-miR-769-3p, hsa-miR-767-5p, hsa-miR-767-3p,hsa-miR-766, hsa-miR-765, hsa-miR-764, hsa-miR-762, hsa-miR-761,hsa-miR-760, hsa-miR-759, hsa-miR-758, hsa-miR-744*, hsa-miR-744,hsa-miR-720, hsa-miR-7-2*, hsa-miR-718, hsa-miR-711, hsa-miR-7-1*,hsa-miR-708*, hsa-miR-708, hsa-miR-7, hsa-miR-675*, hsa-miR-675,hsa-miR-671-5p, hsa-miR-671-3p, hsa-miR-670, hsa-miR-668, hsa-miR-665,hsa-miR-664*, hsa-miR-664, hsa-miR-663b, hsa-miR-663, hsa-miR-662,hsa-miR-661, hsa-miR-660, hsa-miR-659, hsa-miR-658, hsa-miR-657,hsa-miR-656, hsa-miR-655, hsa-miR-654-5p, hsa-miR-654-3p, hsa-miR-653,hsa-miR-652, hsa-miR-651, hsa-miR-650, hsa-miR-649, hsa-miR-648,hsa-miR-647, hsa-miR-646, hsa-miR-645, hsa-miR-644, hsa-miR-643,hsa-miR-642, hsa-miR-641, hsa-miR-640, hsa-miR-639, hsa-miR-638,hsa-miR-637, hsa-miR-636, hsa-miR-635, hsa-miR-634, hsa-miR-633,hsa-miR-632, hsa-miR-631, hsa-miR-630, hsa-miR-629*, hsa-miR-629,hsa-miR-628-5p, hsa-miR-628-3p, hsa-miR-627, hsa-miR-626, hsa-miR-625*,hsa-miR-625, hsa-miR-624*, hsa-miR-624, hsa-miR-623, hsa-miR-622,hsa-miR-621, hsa-miR-620, hsa-miR-619, hsa-miR-618, hsa-miR-617,hsa-miR-616*, hsa-miR-616, hsa-miR-615-5p, hsa-miR-615-3p, hsa-miR-614,hsa-miR-613, hsa-miR-612, hsa-miR-611, hsa-miR-610, hsa-miR-609,hsa-miR-608, hsa-miR-607, hsa-miR-606, hsa-miR-605, hsa-miR-604,hsa-miR-603, hsa-miR-602, hsa-miR-601, hsa-miR-600, hsa-miR-599,hsa-miR-598, hsa-miR-597, hsa-miR-596, hsa-miR-595, hsa-miR-593*,hsa-miR-593, hsa-miR-592, hsa-miR-591, hsa-miR-590-5p, hsa-miR-590-3p,hsa-miR-589*, hsa-miR-589, hsa-miR-588, hsa-miR-587, hsa-miR-586,hsa-miR-585, hsa-miR-584, hsa-miR-583, hsa-miR-582-5p, hsa-miR-582-3p,hsa-miR-581, hsa-miR-580, hsa-miR-579, hsa-miR-578, hsa-miR-577,hsa-miR-576-5p, hsa-miR-576-3p, hsa-miR-575, hsa-miR-574-5p,hsa-miR-574-3p, hsa-miR-573, hsa-miR-572, hsa-miR-571, hsa-miR-570,hsa-miR-569, hsa-miR-568, hsa-miR-567, hsa-miR-566, hsa-miR-564,hsa-miR-563, hsa-miR-562, hsa-miR-561, hsa-miR-559, hsa-miR-558,hsa-miR-557, hsa-miR-556-5p, hsa-miR-556-3p, hsa-miR-555, hsa-miR-554,hsa-miR-553, hsa-miR-552, hsa-miR-551 b*, hsa-miR-551 b, hsa-miR-551 a,hsa-miR-550*, hsa-miR-550, hsa-miR-549, hsa-miR-548q, hsa-miR-548p,hsa-miR-548o, hsa-miR-548n, hsa-miR-548m, hsa-miR-5481, hsa-miR-548k,hsa-miR-548j, hsa-miR-548i, hsa-miR-548h, hsa-miR-548g, hsa-miR-548f,hsa-miR-548e, hsa-miR-548d-5p, hsa-miR-548d-3p, hsa-miR-548c-5p,hsa-miR-548c-3p, hsa-miR-548b-5p, hsa-miR-548b-3p, hsa-miR-548a-5p,hsa-miR-548a-3p, hsa-miR-545*, hsa-miR-545, hsa-miR-544, hsa-miR-543,hsa-miR-542-5p, hsa-miR-542-3p, hsa-miR-541*, hsa-miR-541, hsa-miR-539,hsa-miR-532-5p, hsa-miR-532-3p, hsa-miR-527, hsa-miR-526b*,hsa-miR-526b, hsa-miR-526a, hsa-miR-525-5p, hsa-miR-525-3p,hsa-miR-524-5p, hsa-miR-524-3p, hsa-miR-523*, hsa-miR-523, hsa-miR-522*,hsa-miR-522, hsa-miR-521, hsa-miR-520h, hsa-miR-520g, hsa-miR-520f,hsa-miR-520e, hsa-miR-520d-5p, hsa-miR-520d-3p, hsa-miR-520c-5p,hsa-miR-520c-3p, hsa-miR-520b, hsa-miR-520a-5p, hsa-miR-520a-3p,hsa-miR-519e*, hsa-miR-519e, hsa-miR-519d, hsa-miR-519c-5p,hsa-miR-519c-3p, hsa-miR-519b-5p, hsa-miR-519b-3p, hsa-miR-519a*,hsa-miR-519a, hsa-miR-518f*, hsa-miR-518f, hsa-miR-518e*, hsa-miR-518e,hsa-miR-518d-5p, hsa-miR-518d-3p, hsa-miR-518c*, hsa-miR-518c,hsa-miR-518b, hsa-miR-518a-5p, hsa-miR-518a-3p, hsa-miR-517c,hsa-miR-517b, hsa-miR-517a, hsa-miR-517*, hsa-miR-516b*, hsa-miR-516b,hsa-miR-516a-5p, hsa-miR-516a-3p, hsa-miR-515-5p, hsa-miR-515-3p,hsa-miR-514, hsa-miR-513c, hsa-miR-513b, hsa-miR-513a-5p,hsa-miR-513a-3p, hsa-miR-512-5p, hsa-miR-512-3p, hsa-miR-511,hsa-miR-510, hsa-miR-509-5p, hsa-miR-509-3p, hsa-miR-509-3-5p,hsa-miR-508-5p, hsa-miR-508-3p, hsa-miR-507, hsa-miR-506, hsa-miR-505*,hsa-miR-505, hsa-miR-504, hsa-miR-503, hsa-miR-502-5p, hsa-miR-502-3p,hsa-miR-501-5p, hsa-miR-501-3p, hsa-miR-500*, hsa-miR-500,hsa-miR-499-5p, hsa-miR-499-3p, hsa-miR-498, hsa-miR-497*, hsa-miR-497,hsa-miR-496, hsa-miR-495, hsa-miR-494, hsa-miR-493*, hsa-miR-493,hsa-miR-492, hsa-miR-491-5p, hsa-miR-491-3p, hsa-miR-490-5p,hsa-miR-490-3p, hsa-miR-489, hsa-miR-488*, hsa-miR-488, hsa-miR-487b,hsa-miR-487a, hsa-miR-486-5p, hsa-miR-486-3p, hsa-miR-485-5p,hsa-miR-485-3p, hsa-miR-484, hsa-miR-483-5p, hsa-miR-483-3p,hsa-miR-455-5p, hsa-miR-455-3p, hsa-miR-454*, hsa-miR-454, hsa-miR-453,hsa-miR-452*, hsa-miR-452, hsa-miR-451, hsa-miR-450b-5p,hsa-miR-450b-3p, hsa-miR-450a, hsa-miR-449c*, hsa-miR-449c,hsa-miR-449b*, hsa-miR-449b, hsa-miR-449a, hsa-miR-448, hsa-miR-433,hsa-miR-432*, hsa-miR-432, hsa-miR-431*, hsa-miR-431, hsa-miR-429,hsa-miR-425*, hsa-miR-425, hsa-miR-424*, hsa-miR-424, hsa-miR-423-5p,hsa-miR-423-3p, hsa-miR-422a, hsa-miR-421, hsa-miR-412, hsa-miR-411*,hsa-miR-411, hsa-miR-410, hsa-miR-409-5p, hsa-miR-409-3p, hsa-miR-384,hsa-miR-383, hsa-miR-382, hsa-miR-381, hsa-miR-380*, hsa-miR-380,hsa-miR-379*, hsa-miR-379, hsa-miR-378*, hsa-miR-378, hsa-miR-377*,hsa-miR-377, hsa-miR-376c, hsa-miR-376b, hsa-miR-376a*, hsa-miR-376a,hsa-miR-375, hsa-miR-374b*, hsa-miR-374b, hsa-miR-374a*, hsa-miR-374a,hsa-miR-373*, hsa-miR-373, hsa-miR-372, hsa-miR-371-5p, hsa-miR-371-3p,hsa-miR-370, hsa-miR-369-5p, hsa-miR-369-3p, hsa-miR-367*, hsa-miR-367,hsa-miR-365*, hsa-miR-365, hsa-miR-363*, hsa-miR-363, hsa-miR-362-5p,hsa-miR-362-3p, hsa-miR-361-5p, hsa-miR-361-3p, hsa-miR-34c-5p,hsa-miR-34c-3p, hsa-miR-34b*, hsa-miR-34b, hsa-miR-34a*, hsa-miR-34a,hsa-miR-346, hsa-miR-345, hsa-miR-342-5p, hsa-miR-342-3p, hsa-miR-340*,hsa-miR-340, hsa-miR-33b*, hsa-miR-33b, hsa-miR-33a*, hsa-miR-33a,hsa-miR-339-5p, hsa-miR-339-3p, hsa-miR-338-5p, hsa-miR-338-3p,hsa-miR-337-5p, hsa-miR-337-3p, hsa-miR-335*, hsa-miR-335,hsa-miR-331-5p, hsa-miR-331-3p, hsa-miR-330-5p, hsa-miR-330-3p,hsa-miR-329, hsa-miR-328, hsa-miR-326, hsa-miR-325, hsa-miR-324-5p,hsa-miR-324-3p, hsa-miR-323-5p, hsa-miR-323-3p, hsa-miR-320d,hsa-miR-320c, hsa-miR-320b, hsa-miR-320a, hsa-miR-32*, hsa-miR-32,hsa-miR-31*, hsa-miR-31, hsa-miR-30e*, hsa-miR-30e, hsa-miR-30d*,hsa-miR-30d, hsa-miR-30c-2*, hsa-miR-30c-1*, hsa-miR-30c, hsa-miR-30b*,hsa-miR-30b, hsa-miR-30a*, hsa-miR-30a, hsa-miR-302f, hsa-miR-302e,hsa-miR-302d*, hsa-miR-302d, hsa-miR-302c*, hsa-miR-302c, hsa-miR-302b*,hsa-miR-302b, hsa-miR-302a*, hsa-miR-302a, hsa-miR-301 b, hsa-miR-301 a,hsa-miR-300, hsa-miR-29c*, hsa-miR-29c, hsa-miR-29b-2*, hsa-miR-29b-1*,hsa-miR-29b, hsa-miR-29a*, hsa-miR-29a, hsa-miR-299-5p, hsa-miR-299-3p,hsa-miR-298, hsa-miR-297, hsa-miR-296-5p, hsa-miR-296-3p, hsa-miR-28-5p,hsa-miR-28-3p, hsa-miR-27b*, hsa-miR-27b, hsa-miR-27a*, hsa-miR-27a,hsa-miR-26b*, hsa-miR-26b, hsa-miR-26a-2*, hsa-miR-26a-1*, hsa-miR-26a,hsa-miR-25*, hsa-miR-25, hsa-miR-24-2*, hsa-miR-24-1*, hsa-miR-24,hsa-miR-23b*, hsa-miR-23b, hsa-miR-23a*, hsa-miR-23a, hsa-miR-2278,hsa-miR-2277, hsa-miR-2276, hsa-miR-224*, hsa-miR-224, hsa-miR-223*,hsa-miR-223, hsa-miR-222*, hsa-miR-222, hsa-miR-221*, hsa-miR-221,hsa-miR-220c, hsa-miR-220b, hsa-miR-220a, hsa-miR-22*, hsa-miR-22,hsa-miR-219-5p, hsa-miR-219-2-3p, hsa-miR-219-1-3p, hsa-miR-218-2*,hsa-miR-218-1*, hsa-miR-218, hsa-miR-217, hsa-miR-216b, hsa-miR-216a,hsa-miR-215, hsa-miR-214*, hsa-miR-214, hsa-miR-212, hsa-miR-2117,hsa-miR-2116*, hsa-miR-2116, hsa-miR-2115*, hsa-miR-2115, hsa-miR-2114*,hsa-miR-2114, hsa-miR-2113, hsa-miR-2110, hsa-miR-211, hsa-miR-210,hsa-miR-21*, hsa-miR-21, hsa-miR-20b*, hsa-miR-20b, hsa-miR-20a*,hsa-miR-20a, hsa-miR-208b, hsa-miR-208a, hsa-miR-206, hsa-miR-2054,hsa-miR-2053, hsa-miR-2052, hsa-miR-205*, hsa-miR-205, hsa-miR-204,hsa-miR-203, hsa-miR-202*, hsa-miR-202, hsa-miR-200c*, hsa-miR-200c,hsa-miR-200b*, hsa-miR-200b, hsa-miR-200a*, hsa-miR-200a,hsa-miR-19b-2*, hsa-miR-19b-1*, hsa-miR-19b, hsa-miR-19a*, hsa-miR-19a,hsa-miR-199b-5p, hsa-miR-199b-3p, hsa-miR-199a-5p, hsa-miR-199a-3p,hsa-miR-198, hsa-miR-1979, hsa-miR-1978, hsa-miR-1977, hsa-miR-1976,hsa-miR-1975, hsa-miR-1974, hsa-miR-1973, hsa-miR-1972, hsa-miR-197,hsa-miR-196b*, hsa-miR-196b, hsa-miR-196a*, hsa-miR-196a, hsa-miR-195*,hsa-miR-195, hsa-miR-194*, hsa-miR-194, hsa-miR-193b*, hsa-miR-193b,hsa-miR-193a-5p, hsa-miR-193a-3p, hsa-miR-192*, hsa-miR-192,hsa-miR-1915*, hsa-miR-1915, hsa-miR-1914*, hsa-miR-1914, hsa-miR-1913,hsa-miR-1912, hsa-miR-1911*, hsa-miR-1911, hsa-miR-1910, hsa-miR-191*,hsa-miR-191, hsa-miR-190b, hsa-miR-1909*, hsa-miR-1909, hsa-miR-1908,hsa-miR-190, hsa-miR-18b*, hsa-miR-18b, hsa-miR-18a*, hsa-miR-18a,hsa-miR-188-5p, hsa-miR-188-3p, hsa-miR-187*, hsa-miR-187, hsa-miR-186*,hsa-miR-186, hsa-miR-185*, hsa-miR-185, hsa-miR-184, hsa-miR-183*,hsa-miR-183, hsa-miR-1827, hsa-miR-1826, hsa-miR-1825, hsa-miR-182*,hsa-miR-182, hsa-miR-181d, hsa-miR-181c*, hsa-miR-181 c, hsa-miR-181 b,hsa-miR-181a-2*, hsa-miR-181a*, hsa-miR-181a, hsa-miR-17*, hsa-miR-17,hsa-miR-16-2*, hsa-miR-16-1*, hsa-miR-16, hsa-miR-15b*, hsa-miR-15b,hsa-miR-15a*, hsa-miR-15a, hsa-miR-155*, hsa-miR-155, hsa-miR-154*,hsa-miR-154, hsa-miR-1539, hsa-miR-1538, hsa-miR-1537, hsa-miR-153,hsa-miR-152, hsa-miR-151-5p, hsa-miR-151-3p, hsa-miR-150*, hsa-miR-150,hsa-miR-149*, hsa-miR-149, hsa-miR-148b*, hsa-miR-148b, hsa-miff-148a*,hsa-miR-148a, hsa-miR-147b, hsa-miR-1471, hsa-miR-1470, hsa-miR-147,hsa-miR-146b-5p, hsa-miR-146b-3p, hsa-miR-146a*, hsa-miR-146a,hsa-miR-1469, hsa-miR-1468, hsa-miR-145*, hsa-miR-145, hsa-miR-144*,hsa-miR-144, hsa-miR-143*, hsa-miR-143, hsa-miR-142-5p, hsa-miR-142-3p,hsa-miR-141*, hsa-miR-141, hsa-miR-140-5p, hsa-miR-140-3p,hsa-miR-139-5p, hsa-miR-139-3p, hsa-miR-138-2*, hsa-miR-138-1*,hsa-miR-138, hsa-miR-137, hsa-miR-136*, hsa-miR-136, hsa-miR-135b*,hsa-miR-135b, hsa-miR-135a*, hsa-miR-135a, hsa-miR-134, hsa-miR-133b,hsa-miR-133a, hsa-miR-1324, hsa-miR-1323, hsa-miR-1322, hsa-miR-1321,hsa-miR-132*, hsa-miR-132, hsa-miR-130b*, hsa-miR-130b, hsa-miR-130a*,hsa-miR-130a, hsa-miR-1308, hsa-miR-1307, hsa-miR-1306, hsa-miR-1305,hsa-miR-1304, hsa-miR-1303, hsa-miR-1302, hsa-miR-1301, hsa-miR-1299,hsa-miR-1298, hsa-miR-1297, hsa-miR-1296, hsa-miR-129-5p, hsa-miR-1295,hsa-miR-1294, hsa-miR-129-3p, hsa-miR-1293, hsa-miR-1292, hsa-miR-1291,hsa-miR-1290, hsa-miR-129*, hsa-miR-1289, hsa-miR-1288, hsa-miR-1287,hsa-miR-1286, hsa-miR-1285, hsa-miR-1284, hsa-miR-1283, hsa-miR-1282,hsa-miR-1281, hsa-miR-1280, hsa-miR-128, hsa-miR-1279, hsa-miR-1278,hsa-miR-1277, hsa-miR-1276, hsa-miR-127-5p, hsa-miR-1275, hsa-miR-1274b,hsa-miR-1274a, hsa-miR-127-3p, hsa-miR-1273, hsa-miR-1272, hsa-miR-1271,hsa-miR-1270, hsa-miR-1269, hsa-miR-1268, hsa-miR-1267, hsa-miR-1266,hsa-miR-1265, hsa-miR-1264, hsa-miR-1263, hsa-miR-1262, hsa-miR-1261,hsa-miR-1260, hsa-miR-126*, hsa-miR-126, hsa-miR-125b-2*,hsa-miR-125b-1*, hsa-miR-125b, hsa-miR-125a-5p, hsa-miR-125a-3p,hsa-miR-1259, hsa-miR-1258, hsa-miR-1257, hsa-miR-1256, hsa-miR-1255b,hsa-miR-1255a, hsa-miR-1254, hsa-miR-1253, hsa-miR-1252, hsa-miR-1251,hsa-miR-1250, hsa-miR-1249, hsa-miR-1248, hsa-miR-1247, hsa-miR-1246,hsa-miR-1245, hsa-miR-1244, hsa-miR-1243, hsa-miR-124*, hsa-miR-124,hsa-miR-1238, hsa-miR-1237, hsa-miR-1236, hsa-miR-1234, hsa-miR-1233,hsa-miR-1231, hsa-miR-1229, hsa-miR-1228*, hsa-miR-1228, hsa-miR-1227,hsa-miR-1226*, hsa-miR-1226, hsa-miR-1225-5p, hsa-miR-1225-3p,hsa-miR-1224-5p, hsa-miR-1224-3p, hsa-miR-122*, hsa-miR-122,hsa-miR-1208, hsa-miR-1207-5p, hsa-miR-1207-3p, hsa-miR-1206,hsa-miR-1205, hsa-miR-1204, hsa-miR-1203, hsa-miR-1202, hsa-miR-1201,hsa-miR-1200, hsa-miR-1197, hsa-miR-1185, hsa-miR-1184, hsa-miR-1183,hsa-miR-1182, hsa-miR-1181, hsa-miR-1180, hsa-miR-1179, hsa-miR-1178,hsa-miR-10b*, hsa-miR-10b, hsa-miR-10a*, hsa-miR-10a, hsa-miR-107,hsa-miR-106b*, hsa-miR-106b, hsa-miR-106a*, hsa-miR-106a, hsa-miR-105*,hsa-miR-105, hsa-miR-103-as, hsa-miR-103-2*, hsa-miR-103, hsa-miR-101*,hsa-miR-101, hsa-miR-100*, hsa-miR-100, hsa-miR-1, hsa-let-7i*,hsa-let-7i, hsa-let-7g*, hsa-let-7g, hsa-let-7f-2*, hsa-let-7f-1*,hsa-let-7f, hsa-let-7e*, hsa-let-7e, hsa-let-7d*, hsa-let-7d,hsa-let-7c*, hsa-let-7c, hsa-let-7b*, hsa-let-7b, hsa-let-7a-2*,hsa-let-7a*, hsa-let-7a, hsa-life-1, hsa-life-2, hsa-life-2-AS,hsa-life-3, hsa-life-4, hsa-life-6-5p, hsa-life-6-3p, hsa-life-7-AS,hsa-life-7, hsa-life-9, hsa-life-9-AS, hsa-life-11, hsa-life-12-5p,hsa-life-12-3p, hsa-life-13-3p, hsa-life-13-5p, hsa-life-14-3p,hsa-life-14-5p, hsa-life-17, hsa-life-21, hsa-life-22, hsa-life-26-3p,hsa-life-26-5p, hsa-life-27, hsa-life-31-5p, hsa-life-31-3p,hsa-life-33-AS, hsa-life-33, hsa-life-36-3p, hsa-life-36-5p,hsa-life-37-3p, hsa-life-37-5p, hsa-life-5-5p, hsa-life-5-3p,hsa-life-8, hsa-life-10, hsa-life-15-3p, hsa-life-15-5p, hsa-life-16-5p,hsa-life-16-3p, hsa-life-18, hsa-life-19-5p, hsa-life-19-3p,hsa-life-20-3p, hsa-life-20-5p, hsa-life-23-3p, hsa-life-23-5p,hsa-life-24, hsa-life-25, hsa-life-28-3p, hsa-life-28-5p, hsa-life-29,hsa-life-30, hsa-life-32-AS, hsa-life-32, hsa-life-34-3p,hsa-life-34-5p, hsa-life-35.

3. The method according to item 1 or 2, wherein the predetermined set ofnon-coding RNAs, including miRNAs representative for diagnosis ofprostate cancer comprises at least 1, 7, 10, 15, 20, 25, 30, 35, 40, 50,75, 100 of non-coding RNAs including miRNAs.

4. The method according to item 1, 2 or 3, wherein the predetermined setof miRNAs representative for diagnosis of prostate cancer comprises atleast 1, 7, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100 of the miRNAsselected from the group consisting of hsa-miR-144*, hsa-miR-148a,hsa-miR-519b-5p, hsa-miR-1324, hsa-miR-137, hsa-miR-556-5p,hsa-miR-330-3p, hsa-miR-361-5p, hsa-miR-891 b, hsa-miR-767-5p,hsa-miR-744*, hsa-miR-208b, hsa-miR-548p, hsa-miR-20a*, hsa-miR-195,hsa-miR-33b, hsa-miR-1283, hsa-miR-519c-5p, hsa-miR-497, hsa-miR-9*,hsa-miR-200a, hsa-miR-338-3p, hsa-miR-515-5p, hsa-miR-31*, hsa-miR-551b*, hsa-miR-518e*, hsa-miR-127-5p, hsa-miR-21*, hsa-miR-216a,hsa-miR-452*, hsa-miR-183*, hsa-miR-500, hsa-miR-1826, hsa-miR-625*,hsa-miR-513b, hsa-miR-526a, hsa-miR-33a, hsa-miR-1243, hsa-miR-517*,hsa-miR-541, hsa-miR-217, hsa-miR-621, hsa-miR-518d-5p, hsa-miR-873,hsa-miR-103-as, hsa-miR-450b-5p, hsa-miR-545, hsa-miR-1251,hsa-miR-885-5p, hsa-miR-922, hsa-miR-628-5p, hsa-miR-548f, hsa-miR-802,hsa-miR-25, hsa-miR-423-3p, hsa-miR-522*, hsa-miR-519a*, hsa-miR-455-3p,hsa-miR-1245, hsa-miR-362-5p, hsa-miR-1184, hsa-miR-191, hsa-miR-487a,hsa-miR-216b, hsa-miR-525-5p, hsa-miR-509-3-5p, hsa-miR-27a*,hsa-miR-488*, hsa-miR-1226, hsa-miR-646, hsa-miR-527, hsa-miR-635,hsa-miR-1825, hsa-let-7i*.

5. The method according to any one of items 1-4 wherein said biologicalsample is selected from blood and/or serum or urine samples.

6. The method according to any one of items 1-5 wherein miRNA theexpression profile is determined by nucleic acid hybridization, nucleicacid amplification, polymerase extension, sequencing, mass spectroscopyor any combinations thereof.

7. The method according to any one of items 1-6, wherein the miRNAexpression profile of said subject and the reference expression profilesand optionally the predetermined set of miRNAs are stored in a database.

8. The method according to any one of items 1-7, wherein the biologicalsample is not labeled prior to determination of the expression profile.

9. The method according to any one of items 1-8 wherein the diagnosiscomprises determining survival rate, responsiveness to drugs, and/ormonitoring the course of the disease or the therapy, e.g. chemotherapy.

10. The method of item 6 wherein the nucleic acid hybridisation isperformed using a solid-phase nucleic acid biochip array, in particulara microarray or in situ hybridisation, and/or wherein the nucleic acidamplification is performed via a real-time PCR (RT-PCR).

11. A kit for diagnosing and/or predicting prostate cancer of a subject,comprising:

-   -   (a) means for determining the miRNA expression profile of a RNA        sample of a subject, and    -   (b) at least one reference set of miRNA profile characteristic        for a particular condition.

So far, miRNAs have been extensively studied in tissue material. It hasbeen found that miRNAs are expressed in a highly tissue-specific manner.Disease-specific expression of miRNAs have been reported in many humancancers employing primarily tissue material as the miRNA source. In thiscontext miRNAs expression profiles were found to be useful inidentifying the tissue of origin for cancers of unknown primary origin.Since recently it is known that miRNAs are not only present in tissuesbut also in other body fluid samples, including human blood.Nevertheless, the mechanism why miRNAs are found in body fluids,especially in blood, or their function in these body fluids is notunderstood yet.

Various miRNA biomarkers found in tissue material have been proposed tobe correlated with certain diseases, e.g. cancer. However, there isstill a need for novel miRNAs as biomarkers for the detection and/orprediction of these and other types of diseases. Especially desirableare non-invasive biomarkers, that allow for quick, easy andcost-effective diagnosis/prognosis which cause only minimal stress forthe patient eliminating the need for surgical intervention.

Particularly, the potential role of miRNAs as non-invasive biomarkersfor the diagnosis and/or prognosis of prostate cancer has not beensystematically evaluated yet. In addition, many of the miRNA biomarkerspresently available for diagnosing and/or prognosing of diseases haveshortcomings such as reduced sensitivity, not sufficient specificity ordo not allow timely diagnosis or represent invasive biomarkers.Accordingly, there is still a need for novel and efficient miRNAs orsets of miRNAs as markers, effective methods and kits for thenon-invasive diagnosis and/or prognosis of diseases such as prostatecancer.

The inventors of the present invention assessed for the first time theexpression of miRNAs on a whole-genome level in subjects with prostatecancer as non-invasive biomarkers from body fluids, preferably in blood.They surprisingly found that miRNAs are significantly dysregulated inblood of prostate cancer subjects in comparison to healthy controls andthus, miRNAs are appropriated non-invasive biomarkers for diagnosingand/or prognosing of prostate cancer. This finding is surprising, sincethere is nearly no overlap of the miRNA biomarkers found in blood andthe miRNA biomarkers found in tissue material representing the origin ofthe disease. The inventors of the present invention surprisingly foundmiRNA biomarkers in body fluids, especially in blood, that have not beenfound to be correlated to prostate cancer when tissues material was usedfor this kind of analysis. Therefore, the inventors of the inventionidentified for the first time miRNAs as non-invasive surrogatebiomarkers for diagnosis and/or prognosis of prostate cancer. Theinventors of the present invention identified single miRNAs whichpredict prostate cancer with high specificity, sensitivity and accuracy.The inventors of the present invention also pursued a multiple biomarkerstrategy, thus implementing sets of miRNA biomarkers for diagnosingand/or prognosing of prostate cancer leading to added specificity,sensitivity, accuracy and predictive power, thereby circumventing thelimitations of single biomarker. In detail, by using a machine learningalgorithms, they identified unique sets of miRNAs (miRNA signatures)that allow for non-invasive diagnosis of prostate cancer with evenhigher power, indicating that sets of miRNAs (miRNA signatures) derivedfrom a body fluid sample, such as blood from a subject (e.g. human) canbe used as novel non-invasive biomarkers.

The inventors of the present invention surprisingly found that miRNAsare significantly dysregulated in body fluid samples such as blood ofprostate cancer subjects in comparison to a cohort of controls (healthysubjects) and thus, miRNAs are appropriated biomarkers for diagnosingand/or prognosing of prostate cancer in a non-invasive fashion.Furthermore, the predetermined sets of miRNAs of the present inventionlead to high performance in diagnosing and/or prognosing of prostatecancer, thus expose very high specificity, sensitivity and accuracy.They succeeded in determining the miRNAs that are differentiallyregulated in body fluid samples from patients having prostate cancercompared to a cohort of controls (healthy subjects) (see experimentalsection for experimental details). Additionally, the inventors of thepresent invention performed hypothesis tests (e.g. t-test, limma-test)or other measurements (e.g. AUC, mutual information) on the expressionlevel of the found miRNAs, in all controls (healthy subjects) andsubjects suffering from prostate cancer. These tests resulted in asignificance value (p-value) for each miRNA. This p-value is a measurefor the diagnostic power of each of these single miRNAs to discriminate,for example, between the two clinical conditions: controls (healthysubjects), i.e. not suffering from prostate cancer, or diseased, i.e.suffering from prostate cancer. Since a manifold of tests are carriedout, one for each miRNA, the p-values may be too optimistic and, thus,over-estimate the actual discriminatory power. Hence, the p-values arecorrected for multiple testing by the Benjamini Hochberg approach.

The term “body fluid sample”, as used in the context of the presentinvention, refers to liquids originating from the body of a subject.Said body fluid samples include, but are not limited to, blood, urine,sputum, breast milk, cerebrospinal fluid, cerumen (earwax), endolymph,perilymph, gastric juice, mucus, peritoneal fluid, pleural fluid,saliva, sebum (skin oil), semen, sweat, tears, vaginal secretion, vomitincluding components or fractions thereof. Said body fluid samples maybe mixed or pooled, e.g. a body fluid sample may be a mixture of bloodand urine samples or blood and tissue material. A “body fluid sample”may be provided by removing a body liquid from a subject, but may alsobe provided by using previously isolated sample material. Preferably,the body fluid sample from a subject (e.g. human or animal) has a volumeof between 0.1 and 20 ml, more preferably of between 0.5 and 10 ml, morepreferably between 1 and 8 ml and most preferably between 2 and 5 ml,i.e. 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 2.5, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ml. In thecontext of the present invention said “body fluid sample” allows for anon-invasive diagnosis/and or prognosis of a subject.

The term “blood sample”, as used in the context of the presentinvention, refers to a blood sample originating from a subject. The“blood sample” may be derived by removing blood from a subject byconventional blood collecting techniques, but may also be provided byusing previously isolated and/or stored blood samples. For example ablood sample may be whole blood, plasma, serum, PBMC (peripheral bloodmononuclear cells), blood cellular fractions including red blood cells(erythrocytes), white blood cells (leukocytes), platelets(thrombocytes), or blood collected in blood collection tubes (e.g.EDTA-, heparin-, citrate-, PAXgene- , Tempus-tubes) including componentsor fractions thereof. For example, a blood sample may be taken from asubject suspected to be affected or to be suspected to be affected byprostate cancer, prior to initiation of a therapeutic treatment, duringthe therapeutic treatment and/or after the therapeutic treatment.Preferably, the blood sample from a subject (e.g. human or animal) has avolume of between 0.1 and 20 ml, more preferably of between 0.5 and 10ml, more preferably between 1 and 8 ml and most preferably between 2 and5 ml, i.e. 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 2.5, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ml. In thecontext of the present invention said “body fluid sample” allows for anon-invasive diagnosis/and or prognosis of a subject.

Preferably, when the blood sample is collected from the subject theRNA-fraction, especially the the miRNA fraction, is guarded againstdegradation. For this purpose special collection tubes (e.g. PAXgene RNAtubes from Preanalytix, Tempus Blood RNA tubes from Applied Biosystems)or additives (e.g. RNAlater from Ambion, RNAsin from Promega) thatstabilize the RNA fraction and/or the miRNA fraction are employed.

The biological sample, preferably the body fluid sample may be from asubject (e.g. human or mammal) that has been therapeutically treated orthat has not been therapeutically treated. In one embodiment, thetherapeutical treatment is monitored on the basis of the detection ofthe miRNA or set of miRNAs by the polynucleotide or set ofpolynucleotides of the invention. It is also preferred that total RNA ora subfraction thereof, isolated (e.g. extracted) from a biologicalsample of a subject (e.g. human or animal), is used for detecting themiRNA or set of miRNAs by the polynucleotide or set of polynucleotidesor primer pairs of the invention.

The term “non-invasive”, as used in the context of the presentinvention, refers to methods for obtaining a biological sample,particularly a body fluid sample, without the need for an invasivesurgical intervention or invasive medical procedure. In the context ofthe present invention, a blood drawn represents a non-invasiveprocedure, therefore a blood-based test (utilizing blood or fractionsthereof) is a non-invasive test. Other body fluid samples fornon-invasive tests are e.g. urine, sputum, tears, mothers mild, cerumen,sweat, saliva, vaginal secretion, vomit, etc.

The term “diagnosis” as used in the context of the present inventionrefers to the process of determining a possible disease or disorder andtherefore is a process attempting to define the (clinical) condition ofa subject. The determination of the expression level of a set of miRNAsaccording to the present invention correlates with the (clinical)condition of a subject. Preferably, the diagnosis comprises (i)determining the occurrence/presence of prostate cancer, (ii) monitoringthe course of prostate cancer, (iii) staging of prostate cancer, (iv)measuring the response of a patient with prostate cancer to therapeuticintervention, and/or (v) segmentation of a subject suffering fromprostate cancer.

The term “prognosis” as used in the context of the present inventionrefers to describing the likelihood of the outcome or course of adisease or a disorder. Preferably, the prognosis comprises (i)identifying of a subject who has a risk to develop prostate cancer, (ii)predicting/estimating the occurrence, preferably the severity ofoccurrence of prostate cancer, and/or(iii) predicting the response of asubject with prostate cancer to therapeutic intervention.

The term “suffering or suspected to be suffering from prostate cancer”as used in the context of the present invention comprises the diagnosisand/or prognosis of prostate cancer in a suspect as defined above.

In a first aspect, the present invention relates to a method fordiagnosing and/or prognosing of prostate cancer comprising the steps of:

-   -   (i) determining an expression profile of a predetermined set        comprising at least two miRNAs representative for prostate        cancer in a body fluid sample from a subject, and    -   (ii) comparing said expression profile to a reference expression        profile, wherein the comparison of said expression profile to        said reference expression profile allows for the diagnosis        and/or prognosis of prostate cancer.

It is preferred that the body fluid sample is a blood sample,particularly preferred it is a whole blood, PBMC, serum or plasmasample, more particularly preferred it is a whole blood sample.

It is preferred that the subject is a mammal including both a human andanother mammal, e.g. an animal such as a mouse, a rat, a rabbit, or amonkey. It is particularly preferred that the subject is a human.

Preferably, the predetermined set comprising at least two miRNAs isselected from the set of miRNAs listed in FIG. 2 or 5.

It is preferred that the predetermined set comprising at least twomiRNAs is selected from the sets of miRNAs listed in FIG. 6 (SNP-1 toSNP-911). It is also preferred that the predetermined set comprising atleast two miRNAs comprises at least one set of miRNAs listed in FIG. 6.

Further, in a preferred embodiment of the method of the presentinvention, for determining an expression profile of the predeterminedset comprising at least two miRNAs representative for prostate cancer ina body fluid sample from a subject comprises the miRNAs from one set ora plurality of sets of miRNAs listed in FIG. 6.

For example, a set comprising 30 miRNAs representative for prostatecancer in a body fluid sample from a subject comprises at least themiRNAs from one predetermined set or several sets of miRNAs listed inFIG. 6. Alternatively, a set comprising 29, 28, 27,26, 25, 24, 23, 22,21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3miRNAs representative for prostate cancer comprises at least the miRNAsfrom one set or several sets of miRNAs listed in FIG. 6.

Further, in another preferred embodiment of the method of the presentinvention, for determining an expression profile of the predeterminedset comprising at least two miRNAs representative for prostate cancer ina body fluid sample from a subject comprises combinations of sets ofmiRNAs listed in FIG. 6.

For example, said predetermined set comprising 30 miRNAs representativefor prostate cancer in a body fluid sample from a subject comprises atleast 2, e.g. 2, 3, 4, 5 or 6, sets of miRNAs listed in FIG. 6.Alternatively, said set comprising 29, 28, 27, 26, 25, 24, 23, 22, 21,20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or 4 miRNAscomprises a least 2, e.g. 2, 3, 4, 5 or 6, sets of miRNAs listed in FIG.6.

The reference expression profile may be obtained from at least twosubjects (e.g. human or animal). Preferably the reference expressionprofile is an average expression profile (data) of at least 2 to 400subjects, more preferably at least 20 to 200 subjects, and mostpreferably at least 40 to 150 subjects, with one known clinicalcondition which is prostate cancer or a specific form of prostatecancer.

It is particularly preferred that the reference expression profile is analgorithm or mathematical function. Preferably the algorithm ormathematical function is obtained from a reference expression profile(data) of at least two subjects, preferably the algorithm ormathematical function is obtained from an average reference expressionprofile (data) of at least 2 to 400 subjects, more preferably of atleast 20 to 200 subjects, and most preferably of at least 40 to 150subjects.

It is preferred that the algorithm or mathematical function is obtainedusing a machine learning approach.

Preferably, the algorithm or mathematical function is saved on a datacarrier comprised in the kit (according to the seventh aspect of theinvention) or the computer program, wherein the algorithm ormathematical function is comprised, is saved on a data carrier comprisedin the kit.

It is preferred that the miRNA expression profile may be generated byany convenient means, e.g. nucleic acid hybridization (e.g. to amicroarray), nucleic acid amplification (PCR, RT-PCR, qRT-PCR,high-throughput RT-PCR), ELISA for quantitation, next generationsequencing (e.g. ABI SOLID, Illumina Genome Analyzer, Roche/454 GS FLX),flow cytometry (e.g. LUMINEX) and the like, that allow the analysis ofdifferential miRNA expression levels between samples of a subject (e.g.diseased) and a control subject (e.g. healthy, reference sample).

Nucleic acid hybridization may be performed using a microarray/biochipor in situ hybridization. In situ hybridization is preferred for theanalysis of a single miRNA or a set comprising a low number of miRNAs(e.g. a set of at least 2 to 50 miRNAs such as a set of 2, 5, 10, 20,30, or 40 miRNAs). The microarray/biochip, however, allows the analysisof a single miRNA as well as a complex set of miRNAs (e.g. a all knownmiRNAs or subsets therof).

Nucleic acid amplification may be performed using real time polymerasechain reaction (RT-PCR) such as real time quantitative polymerase chainreaction (RT qPCR). The standard real time polymerase chain reaction(RT-PCR) is preferred for the analysis of a single miRNA or a setcomprising a low number of miRNAs (e.g. a set of at least 2 to 50 miRNAssuch as a set of 2, 5, 10, 20, 30, or 40 miRNAs), whereashigh-throughput RT-PCR technologies (e.g. OpenArray from AppliedBiosystems, SmartPCR from Wafergen, Biomark System from Fluidigm) arealso able to measure large sets of miRNAS (e.g. a set of 10, 20, 30, 50,80, 100, 200 or more) or all known miRNAs in a high parallel fashion.RT-PCR is particularly suitable for detecting low abandoned miRNAs.

In a second aspect, the invention relates to a set comprisingpolynucleotides for detecting a predetermined set comprising at leasttwo miRNAs for diagnosing and/or prognosing of prostate cancer in a bodyfluid sample from a subject.

It is preferred that the body fluid sample is a blood sample,particularly preferred it is a whole blood, PBMC, serum or plasmasample, more particularly preferred it is a whole blood sample.

It is preferred that the subject is a mammal including both a human andanother mammal, e.g. an animal such as a mouse, a rat, a rabbit, or amonkey. It is particularly preferred that the subject is a human.

Preferably, the predetermined set comprising at least two miRNAs isselected from the set of miRNAs listed in FIG. 2 or 5.

It is preferred that the predetermined set comprising at least twomiRNAs is selected from the set of miRNAs listed in FIG. 6. It ispreferred that the predetermined set comprising at least two miRNAscomprises at least one set of miRNAs listed in FIG. 6.

It is preferred that the polynucleotides comprised in the set of thepresent invention are complementary to the miRNAs comprised in thepredetermined set, wherein the nucleotide sequences of said miRNAs arepreferably selected from the group consisting of miRNAs listed in FIG. 2or 5 or set of miRNAs listed in FIG. 6, a fragment thereof, and asequence having at least 80%, 85%, 90% or 95% sequence identity thereto.

For example, the polynucleotides of the present invention are fordetecting a predetermined set of 40 or 39 or 38 or 37 or 36 or 35 or 34or 33 or 32 or 31 or 30 or 29 or 28 or 27 or 26 or 25 or 24 or 23 or 22or 21 or 20 or 19 or 18 or 17 or 16 or 15 or 14 or 13 or 12 or 11 or 10or 9 or 8 or 7 or 6 or 5 or 4 or 3 miRNAs wherein the set of miRNAscomprises at least one, e.g. 1, 2, 3, 4, 5 or 6, of the set of miRNAslisted in FIG. 6.

In a third aspect, the invention relates to the use of set ofpolynucleotides according to the second aspect of the invention fordiagnosing and/or prognosing prostate cancer in a subject.

In a fourth aspect, the invention relates to a set of at least twoprimer pairs for determining the expression level of a predetermined setof miRNAs in a body fluid sample of a subject suffering or suspected ofsuffering from prostate cancer.

It is preferred that the body fluid sample is a blood sample,particularly preferred it is a whole blood, PBMC, serum or plasmasample, more particularly preferred it is a whole blood sample.

It is preferred that the subject is a mammal including both a human andanother mammal, e.g. an animal such as a mouse, a rat, a rabbit, or amonkey. It is particularly preferred that the subject is a human.

Preferably, the predetermined set comprising at least two miRNAs isselected from the set of miRNAs listed in FIG. 2 or 5.

It is preferred that the predetermined set comprising at least twomiRNAs is selected from the sets of miRNAs listed in FIG. 6. It ispreferred that the predetermined set comprising at least two miRNAscomprises at least one set of miRNAs listed in FIG. 6.

It is preferred that the, set of at least two primer pairs fordetermining the expression level of a predetermined set of miRNAs in abody fluid sample of a subject suffering or suspected of suffering fromprostate cancer are primer pairs that are specific for at least onemiRNA listed in FIG. 2 or 5.

It is preferred that the set of at least two primer pairs fordetermining the expression level of a predetermined set of miRNAs in abody fluid sample of a subject suffering or suspected of suffering fromprostate cancer are primer pairs that are specific for at least one setof miRNAs listed in FIG. 6.

It is preferred that the set of at least two primer pairs of the presentinvention are for detecting a set comprising, essentially consisting of,or consisting of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40or more miRNAs, and wherein the set of miRNAs comprises at least one ofthe sets listed in FIG. 6.

For example, the set of at least two primer pairs of the presentinvention are for detecting a predetermined set of 40 or 39 or 38 or 37or 36 or 35 or 34 or 33 or 32 or 31 or 30 or 29 or 28 or 27 or 26 or 25or 24 or 23 or 22 or 21 or 20 or 19 or 18 or 17 or 16 or 15 or 14 or 13or 12 or 11 or 10 or 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 miRNAs whereinthe predetermined set of miRNAs comprises at least one of the set ofmiRNAs listed in FIG. 6.

Preferably, the said primer pairs may be used for amplifying cDNAtranscripts of the predetermined set of miRNAs selected from the miRNAslisted in FIG. 2 or FIG. 5. Furthermore, the said primer pairs may beused for amplifying cDNA transcripts of the set of miRNAs listed in FIG.6.

It is understood that the primer pairs for detecting a predetermined setof miRNAs may consist of specific and or non-specific primers.Additionally, the set of primer pairs may be complemented by othersubstances or reagents (e.g. buffers, enzymes, dye, labelled probes)known to the skilled in the art for conducting real time polymerasechain reaction (RT-PCR).

In a fifth aspect, the invention relates to the use of a set of primerpairs according to the fourth aspect of the invention for diagnosingand/or prognosing prostate cancer in a subject.

In a sixth aspect, the invention relates to means for diagnosing and/orprognosing of prostate cancer in a body fluid sample of a subject.

Preferably, the invention relates to means for diagnosing and/orprognosing of prostate cancer in a body fluid sample of a subjectcomprising

-   -   (i) a set of at least two polynucleotides according to the        second aspect of the invention or    -   (ii) a set of at least two primer pairs according the fourth        aspect of the invention.

It is preferred that the body fluid sample is a blood sample,particularly preferred it is a whole blood, PBMC, serum or plasmasample, more particularly preferred it is a whole blood sample.

It is preferred that the subject is a mammal including both a human andanother mammal, e.g. an animal such as a mouse, a rat, a rabbit, or amonkey. It is particularly preferred that the subject is a human.

Preferably, that the set of at least two polynucleotides or the set ofat least 2 primer pairs are for detecting a predetermined set comprisingat least two miRNAs for diagnosing and/or prognosing of prostate cancerin a body fluid sample, e.g. blood sample, from a subject, e.g. patient,human or animal, wherein the set of miRNAs is selected from the miRNAslisted in FIG. 2 or FIG. 5.

It is preferred that the set of at least two polynucleotides or the setof at least 2 primer pairs are for detecting a predetermined setcomprising at least two miRNAs for diagnosing and/or prognosing ofprostate cancer in a body fluid sample, e.g. blood sample, from asubject, e.g. patient, human or animal, wherein the set of miRNAs isselected from the sets of miRNAs listed in FIG. 6.

It is preferred that the set of at least two primer pairs fordetermining the expression level of a predetermined set of miRNAs in abody fluid sample of a subject suffering or suspected of suffering fromprostate cancer are primer pairs that are specific for at least twomiRNAs selected from the miRNAs listed in FIG. 2 or FIG. 5.

It is preferred that the set of at least two primer pairs fordetermining the expression level of a set of miRNAs in a body fluidsample of a subject suffering or suspected of suffering from prostatecancer are primer pairs that are specific for at least one set of miRNAslisted in FIG. 6.

It is also preferred that said means for diagnosing and/or prognosing ofprostate cancer comprise, of a set of beads comprising a at least twopolynucleotides according to the second aspect of the present invention.It is especially preferred that the beads are employed within a flowcytometer setup for diagnosing and/or prognosing of prostate cancer,e.g. in a LUMINEX system (www.luminexcorp.com).

In a seventh aspect, the invention relates to a kit for diagnosingand/or prognosing of prostate cancer in a subject.

Preferably, the invention relates to a kit for diagnosing and/orprognosing of prostate cancer comprising

-   -   (i) means for determining an expression profile of a        predetermined set comprising at least two miRNAs representative        for prostate cancer in a body fluid sample from a subject, and    -   (ii) at least one reference.

It is preferred that the body fluid sample is a blood sample,particularly preferred it is a whole blood, PBMC, serum or plasmasample, more particularly preferred it is a whole blood sample.

It is preferred that the subject is a mammal including both a human andanother mammal, e.g. an animal such as a mouse, a rat, a rabbit, or amonkey. It is particularly preferred that the subject is a human.

Said means may comprise of at least two polynucleotides according to thesecond aspect of the present invention, a set of at least 2 primer pairsaccording to the fourth aspect of the invention; means according to thesixth aspect of the present invention; primers suitable to performreverse transcriptase reaction and/or real time polymerase chainreaction such as quantitative polymerase chain reaction; and/or meansfor conducting next generation sequencing.

In an eighth aspect, the invention relates to a predetermined set ofmiRNAs in a body fluid sample isolated from a subject for diagnosingand/or prognosing of prostate cancer.

It is preferred that the body fluid sample is a blood sample,particularly preferred it is a whole blood, PBMC, serum or plasmasample, more particularly preferred it is a whole blood sample.

It is preferred that the subject is a mammal including both a human andanother mammal, e.g. an animal such as a mouse, a rat, a rabbit, or amonkey. It is particularly preferred that the subject is a human.

Preferably, the predetermined set comprising at least two miRNAs isselected from the set of miRNAs listed in FIG. 2 or 5.

It is preferred that the predetermined set comprising at least twomiRNAs is selected from the set of miRNAs listed in FIG. 6. It ispreferred that the predetermined set comprising at least two miRNAscomprises at least one set of miRNAs listed in FIG. 6.

In a ninth aspect, the invention relates to the use of a set of miRNAsaccording to the eighth aspect of the invention for diagnosing and/orprognosing of prostate cancer in a subject.

The invention will now be illustrated by the following figures and thenon-limiting experimental examples.

FIGURES

FIG. 1:

Overview of miRNA sequences published in the miRNA database 14.0 plusadditional miRNA sequences.

FIG. 2:

Overview of all miRNAs that are found to be differentially regulated inblood samples of prostate cancer patients, grouped accordingly to theirresults in t-tests.

FIG. 3:

General overview of the method of diagnosing and/or predicting the stateof health employing predetermined sets of miRNAs.

FIG. 4:

Prostate cancer patients against healthy controls - classification of:according to t-test with the 270 miRNAs with the lowest p-values (seeFIG. 2) lead to an accuracy 82.8% a specificity of 87.5% and asensitivity of 71.9%

red=prostate cancer patients (1=derived from 1 independent samplecollection); blue=healthy controls (1,2,3,4,5=derived from 5 independentsample collections)

FIG. 5:

Overview of miRNAs that are found to be differentially regulated betweenhealthy control and subjects suffering from prostate cancer.Experimental details: SEQ ID NO: sequence identification number, miRNA:identifier of the miRNA according to miRBase, median g1: medianintensity obtained from microarray analysis for healthy controls, mediang2: median intensity obtained from microarray analysis for individualswith prostate cancer, qmedian: ratio of median g1/median g2, logqmedian:log of qmedian, ttest_rawp: p-value obtained when applying t-test,ttest_adjp: adjusted p-value in order to reduce false discovery rate byBenjamini-Hochberg adjustment, AUC: Area under the curve, limma_rawp:p-value obtained when applying limma-test, limma_adjp: adjusted p-valuein order to reduce false discovery rate by Benjamini-Hochbergadjustment.

FIG. 6:

Predetermined sets of miRNAs (miRNA signatures SNP-1 to 911) that allowfor effective diagnosis and/or prognosis of subjects suffering orsubjects suspected to suffering from prostate cancer. Experimentaldetails: SEQ ID NO: sequence identification number, miRNA: identifier ofthe miRNA according to miRBase, Acc=accuracy, Spec=specificity,Sens=sensitivity

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1. A method of diagnosing prostate cancer, comprising the steps (a)determining an expression profile of a predetermined set of miRNAs, in ablood sample from a patient, particularly a human patient; and (b)comparing said expression profile to a reference expression profile,wherein the comparison of said determined expression profile to saidreference expression profile allows for the diagnosis of prostatecancer.
 2. The method according to claim 1, wherein the expressionprofile is determined from miRNAs selected from FIG. 2 or FIG. 5
 3. Themethod according to claim 1, wherein the predetermined set of miRNAscomprises at least one set of miRNAs listed in FIG.
 6. 4. The methodaccording to claim 1, wherein the predetermined set of miRNAsrepresentative for diagnosis of prostate cancer comprises at least 1, 7,10, 15, 20, 25, 30, 35, 40, 50, 75, 100 of miRNAs.
 5. The methodaccording to any claim 1 wherein the expression profile is determined bynucleic acid hybridization, nucleic acid amplification, polymeraseextension, sequencing, mass spectroscopy, flow cytometry or anycombinations thereof.
 6. A set of polynucleotides for detecting apredetermined set comprising at least two miRNAs for diagnosing and/orprognosing of prostate cancer in a blood sample from a patient,particularly a human patient.
 7. The set of polynucleotides according toclaim 6, wherein the miRNAs are selected from the miRNAs listed in FIG.2 or
 5. 8. The set of polynucleotides claim 6, wherein the predeterminedset of miRNAs is selected from the sets of miRNAs listed in FIG.
 6. 9.The set of polynucleotides according to claim 6, wherein thepredetermined set of miRNAs representative for diagnosis of prostatecancer comprises at least 1, 7, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100of miRNAs.
 10. Use of set of polynucleotides according to claim 6 fordiagnosing and/or prognosing prostate cancer in a patient, particularlya human patient.
 11. A set of primer pairs for determining theexpression level of a predetermined set of miRNAs in a blood sample of apatient for diagnosing and/or prognosing prostate cancer.
 12. The set ofprimer pairs according to claim 11, wherein the miRNAs are selected fromthe miRNAs listed in FIG. 2 or FIG.
 5. 13. The set of primer pairsaccording to claim 11, wherein the predetermined set of miRNAs comprisesat least one set of miRNAs listed in FIG.
 6. 14. The set of primer pairsaccording to claim 11, wherein the predetermined set of miRNAsrepresentative for diagnosis of prostate cancer comprises at least 1, 7,10, 15, 20, 25, 30, 35, 40, 50, 75, 100 of miRNAs.
 15. Use of set ofprimer pairs according to claim 11 for diagnosing and/or prognosingprostate cancer in a patient, particularly a human patient.
 16. Meansfor diagnosing and/or prognosing of prostate cancer in a blood sample ofa subject comprising : a set of at least two polynucleotides accordingto claim 6 or a set of at least two primer pairs capable of determiningthe expression level of a predetermined set of miRNAs in a blood sample.17. A kit for diagnosing and/or predicting prostate cancer, comprising:(a) means for determining the miRNA expression profile of a RNA sampleof a subject, and (b) at least one reference expression profile for aparticular condition.
 18. The kit according to claim 17 comprising a setof polynucleotides capable of detecting a predetermined set comprisingat least two miRNAs in a blood sample, or a set of at least two primerpairs capable of determining the expression level of a predetermined setof miRNAs in a blood sample.
 19. A set of miRNAs isolated from a bloodsample from a subject for diagnosing and/or prognosing of prostatecancer, wherein the miRNAs are selected from the miRNAs as indicated inclaim
 2. 20. The set of miRNAs of claim 19 bound to a carrier, e.g. amicroarray.
 21. Use of a set of miRNAs according to claim 19 fordiagnosing and/or prognosing of prostate cancer in a subject,particularly a human subject.
 22. The method of claim 1, wherein saidsample is a whole blood sample.